Esterases and lipases are hydrolases which can be employed in industrial processes for synthesizing optically active organic compounds and which are characterized by high substrate specificity. Through a mechanism similar to that of serine proteases, they can transfer acyl groups onto nucleophiles such as, for example, carbonyl groups or hydrolytically cleave ester bonds. Esterases, lipases and serine proteases share the catalytic triad, a sequence motif consisting of the amino acids Ser, His and Asp, where the carbonyl carbon atom is subject to nucleophilic attack by the active Ser, which, with participation of the other two amino acids, leads to a charge distribution. Esterases and lipases may also transfer acyl groups onto other nucleophiles, such as thioether thio groups or activated amines.
Lipases hydrolyze long-chain glycerol esters and are characterized by surface activation, i.e. the active site becomes accessible only in the presence of the lipid substrate. Lipases are stable in nonaqueous organic solvents and are employed in numerous industrial processes for kinetic racemate resolution, i.e. one enantiomer is converted substantially faster than the other. Said enantiomer can be subsequently obtained from the reaction solution owing to different physical and chemical properties.
Nakamura (Nakamura, K. et al., Tetrahedron; Asymmetry 9, (1999), 4429-4439) describes the racemate resolution of 1-alkyn-3-ol by transesterification in hydrophobic solvents with the aid of commercially available lipases (Amano AK, AH and PS; Amano Pharmaceuticals Co. Ltd.). In this reaction, enantioselectivity increases with the chain length of the acyl donor and sterically large residues (chloroacetate, vinylbenzoate) have an adverse effect on the reaction. Yang (Yang, H. et al., J. Org. Chem. 64, (1999), 1709-1712) describes the enantioselective preparation of optically active acids by transesterification with vinyl esters using lipase B from Candida antarctica as catalyst. In this case, ethyl esters lead to a distinctly lower reaction rate and selectivity. A lipase isolated from Burkholderia plantarii (Pseudomonas plantarii or glumae) DSM 6535 is employed for enantioselective acylation of racemic amines with the aid of ethyl methoxy-acetate (Balkenhohl, F. et al., J. prakt. Chem. 339, (1997), 381-384).
Esterases enantioselectively catalyze the formation and breaking of ester bonds (forward and reverse reaction). Preference is given to using vinyl esters in the transesterification for obtaining optically active alcohols, since the alcohol function of the ester is no longer available after the conversion due to tautomerization to the aldehyde or ketone and thus the reverse reaction can be avoided. In contrast to lipases, esterases are not surface-activated and also convert organic compounds of relatively short chain length. Esterases of different substrate specificity have been isolated from various organisms.
Thus the esterase from Pseudocardia thermophila FERM-BP-6275 is used for hydrolyzing optically active chromanacetic esters (EP-A-0 892 044).
An esterase from Bacillus acidocaldarius hydrolyzes with low enantioselectivity esters from a narrow range of substrates (Manco, G. et al., Biochem. J. 332, (1998), 203-212).
Acylase 1 from Aspergillus is used for obtaining secondary alcohols by transesterification with vinyl esters in organic nonpolar solvents, it being preferred to convert secondary alcohols having short side chains (Faraldos, J. et al., Synlett 4, (1997), 367-370). From Pseudomonas fluorescens DSM 50 106 a membrane-bound lactone-specific esterase has been cloned (Khalameyzer, V. et al., Appl. and Environ. Microbiol. 65(2), (1999), 477-482), and from the E. coli malQ mutant an acetylesterase has been cloned (Peist, R. et al., J. Bacteriol. 179, (1997), 7679-7686). However, enantioselectivity and substrate specificity of these two esterases have not been studied in more detail. Rhodococcus sp. NCBM 11216 expresses 4 esterases, RR1 to RR4, which have different specificity. For the ester synthesis from naphthol and an acid, RR1 and RR2 prefer acids with short carbon chains, while RR3 and RR4 specifically convert acids having relatively long carbon chains and sterically relatively large residues (Gudelj, M. et al., J. Mol. Cat. B, Enzymatic 5, (1998), 261-266).
However, esterases which have a wide range of substrates and a high enantioselectivity and which can be employed in industrial processes are not available for preparing small organic molecules, such as optically active alcohols, acids or esters with short carbon chains.